Interrelationship among annual cycles of sex steroids, corticosterone and body condition in Nyctibatrachus humayuni.

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Does physiological response to disease incur cost to reproductive ecology in a sexually dichromatic amphibian species?

It is well known that the disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has contributed to amphibian declines worldwide. The impact of Bd varies, with some species being more susceptible to infection than others. Recent evidence has shown that Bd can have sub-lethal effects, whereby increases in stress hormones have been associated with infection. Could this increased stress response, which is a physiological adaptation that provides an increased resilience against Bd infection, potentially be a trade-off with important life-history traits such as reproduction? We studied this question in adult male frogs of a non-declining species (Litoria wilcoxii). Frogs were sampled for (1) seasonal hormone (testosterone and corticosterone), color and disease profiles, (2) the relationship between disease infection status and hormone levels or dorsal color, (3) subclinical effects of Bd by investigating disease load and hormone level, and (4) reproductive and stress hormone relationships independent of disease. Testosterone levels and color score varied seasonally (throughout the spring/summer months) while corticosterone levels remained stable. Frogs with high Bd prevalence had significantly higher corticosterone levels and lower testosterone levels compared to uninfected frogs, and no differences in color were observed. There was a significant positive correlation between disease load and corticosterone levels, and a significant negative relationship between disease load and testosterone. Our field data provides novel evidence that increased physiological stress response associated with Bd infection in wild frogs, could suppress reproduction by down-regulating gonadal hormones in amphibians, however the impacts on reproductive output is yet to be established.

Non-invasive monitoring of glucocorticoid physiology within highland and lowland populations of native Australian Great Barred Frog (Mixophyes fasciolatus).

This study used non-invasive endocrinology to examine baseline corticosterone at different altitudes in a free-living Australian amphibian: the Great Barred Frog (Mixophyes fasciolatus). An enzyme immunoassay (EIA) was performed on urine samples and validated biologically using an adrenocorticotropic hormone (ACTH) challenge. Frogs were injected with ACTH on day 0 and recaptures occurred 1-10days post injection. Urine samples and body condition measurements were collected from lowland (60m) and highland (660m and 790m) sub-populations of M. fasciolatus in South East Queensland (SEQ), close to their post-breeding period during autumn 2011. We simultaneously sampled these sub-populations for Batrachochytrium dendrobatidis (Bd), a pathogenic fungus responsible for mass mortalities of amphibians worldwide. The ACTH challenge successfully validated the urinary corticosterone EIA in M. fasciolatus, with a peak urinary corticosterone response to ACTH injection on day 2 and a return to baseline levels by day 6. Polymerase chain reaction (PCR) analysis of 50 individuals returned only 1 positive result for Bd. Simple linear regression showed a strong positive relationship between baseline urinary corticosterone concentrations and altitude and no relationship with body condition. We hypothesize that higher baseline corticosterone concentrations within highland sub-populations of male M. fasciolatus could be associated with increased environmental challenge at high altitudes and geographic range limits. Whether this pattern is an indication of chronic stress in highland populations or life-time fitness and survival, warrants future investigation.

Repeatability of baseline corticosterone and short-term corticosterone stress responses, and their correlation with testosterone and body condition in a terrestrial breeding anuran (Platymantis vitiana).

Repeatability of physiological response variables, such as the stress hormone corticosterone, across numerous sampling occasions is an important assumption for their use as predictors of behaviour, reproduction and fitness in animals. Very few studies have actually tested this assumption in free-living animals under uncontrolled natural conditions. Non-invasive urine sampling and standard capture handling protocol have enabled the rapid quantification of baseline corticosterone and short-term corticosterone stress responses in anuran amphibians. In this study, established non-invasive methods were used to monitor physiological stress and urinary testosterone levels in male individuals of the terrestrial breeding Fijian ground frog (Platymantis vitiana). Adult male frogs (n = 20) were sampled at nighttime on three repeated occasions at intervals of 14 days during their annual breeding season on Viwa Island, Fiji. All frogs expressed urinary corticosterone metabolite responses to the capture and handling stressor, with some frogs showing consistently higher urinary corticosterone responses than others. Ranks of corticosterone values at 0, 4 and 8 h, and the corrected rank were highly significant (r = 0.75-0.99) between the three repeated sampling occasions. Statistical repeatabilities were high for baseline corticosterone (r = 0.973) and for corticosterone values at 2 h (r = 0.862), 4 h (r = 0.861), 6 h (r = 0.820) and 8 h (r = 0.926), and also for the total (inclusive of baseline corticosterone values) and the corrected integrated responses (index of the acute response) [r = 0.867 and r = 0.870]. Urinary testosterone levels also showed high statistical repeatability (r = 0.78). Furthermore, variation in baseline and short-term corticosterone stress responses was greater between individuals than within individuals. Baseline urinary corticosterone was significantly negatively correlated with the corrected integrated corticosterone response (r = -0.3, p < 0.001) but non-significantly with body-condition (r = -0.04) and baseline urinary testosterone (r = -0.07). In contrast, the corrected integrated corticosterone response was positively correlated (non-significantly) with baseline urinary testosterone (r = +0.04) and body-condition (r = +0.08). Urinary testosterone levels and body-condition were significantly negatively correlated (r = -0.23, p < 0.001). The results suggest that male frogs with higher levels of testosterone could have depleted energy reserve during the breeding period. The acute corticosterone responses help in replenishing energy that is needed for breeding and survival. The results also provide some support to the 'cort-fitness' hypothesis as highlighted by the negative correlation between baseline corticosterone and body-condition. It is most likely that the acute corticosterone response is adaptive and linked positively with reproductive fitness and survival in male anurans.

The effect of stress and stress hormones on dynamic colour-change in a sexually dichromatic Australian frog.

Rapid colour changes in vertebrates have fascinated biologists for centuries, herein we demonstrate dynamic colour change in an anuran amphibian, the stony creek frog (Litoria wilcoxii), which turns from brown to bright (lemon) yellow during amplexus. We show this by comparing the colour of baseline (unpaired males) and amplecting (paired) males. We also investigate the possible role of stress and stress hormones on this colour change. Frogs were subjected to four different levels of stressors (handling, toe-clipping, saline injection and adrenocorticotropic hormone [ACTH] injection) and the colour change was measured using digital photography. A comparison of baseline colour and stress hormone (corticosterone) levels was also conducted to give further insight to this topic. From the images, the Red Blue Green (RGB) colour values were calculated, and a principal components analysis (PCA) was used to create a single colour metric (the major axis) as an index of colour in the visible spectrum. A moderate stressor (toe-clipping) led to a significant change in colour (within 10 min) similar to that of amplecting males. Surprisingly, neither a mild stressor (handling and saline injection) nor the maximum stressor (handling and ACTH injection) led to a lightening response. This study confirms that the dynamic male colour change in this species in response to medium stressors adds new knowledge to the understanding of the functional mechanisms of dynamic colour change in amphibians.

Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n=20) immediately upon field capture. Toads were handled for 5min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8h in the field. The toads were resampled for baseline (0h) urine corticosterone with hourly urine sampling over 8h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r=0.877) together with high statistical repeatabilities for 3h (r=0.695), 6h (r=0.428) and 8h (r=0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.807; r=0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.

Urinary corticosterone metabolite responses to capture and handling in two closely related species of free-living Fijian frogs.

Studies of baseline (unstressed) and short-term corticosterone stress responses in free-living amphibians can provide crucial information on the physiological responses of different populations to environmental change. In this study, we compared baseline and urinary corticosterone metabolite responses of free-living adult males and females of two closely related Fijian frogs of the Platymantis genus (Family: Ceratobatrachidae). Fijian ground frogs (Platymantis vitiana) live on the ground while Fijian tree frogs (Platymantis vitiensis) are arboreal. We captured free-living frogs and applied our moderate stress protocol (5 min handling during urine sampling at hourly intervals), with urinary corticosterone metabolite concentrations measured by enzyme-immunoassay. Mean urinary corticosterone metabolite concentrations in male and female Fijian ground frogs increased from 0 to 2 h and continued to increase to peak concentrations 5-6 h after capture. Mean baseline corticosterone concentration was significantly different between sexes (higher in males than females) only for Fijian ground frogs. There was no significant difference between sexes in the integrated corticosterone responses for both species. Mean baseline and urinary corticosterone metabolite responses of Fijian tree frogs were lower than those of Fijian ground frogs. Corticosterone levels increased for 4-5 h in both species and began to decrease again 7 h after initial capture. Corticosterone responses were consistently higher for Fijian ground frogs than Fijian tree frogs. Individuals in both species showed markedly variable corticosterone responses over the 8h duration of the stressor, with some individuals showing low stress responses and others showing high stress responses. The magnitude of the corrected integrated response of the ground frogs was almost twice that of the tree frogs. These differences in baseline and short-term corticosterone stress responses between these two species could be a consequence of ecological differences including micro-habitat, predator interactions and/or competitive interactions with the introduced cane toad (Rhinella marina). Comparisons of corticosterone responses between populations and species provide a valuable tool for measuring the physiological responses of the amphibians to environmental change.

Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii).

The emerging amphibian disease chytridiomycosis, which is caused by the fungal pathogen (Batrachochytrium dendrobatidis, Bd), has caused mass mortalities of native amphibian populations globally. There have been no previous studies on the relationships between stress hormones in free-living amphibians and Bd infections. In this study, we measured urinary corticosterone metabolite concentrations and Bd infections within free-living populations of male Stony Creek frog (Litoria wilcoxii) in Queensland, Australia. Prevalence of Bd zoospores from frog skin swabs was quantified using a real-time quantitative PCR technique. A urinary corticosterone enzyme-immunoassay (EIA) was validated using adrenocorticotropic hormone (ACTH) challenge. Urinary corticosterone concentrations of male frogs increased within 1-2 days after ACTH challenge and returned to baseline levels within 3 days post-ACTH injection. None of the frogs showed any rise in urinary corticosterone after saline injections. Individual male frogs showed either low or high baseline corticosterone concentrations. Male frogs identified as positive for Bd infection had significantly higher baseline urinary corticosterone concentrations in comparison to Bd negative male frogs. Urinary corticosterone EIA provides a reliable indication of stress in this frog species and this non-invasive physiological tool can be used to further assess the dynamics of Bd infections and physiological stress responses in other native amphibians.

Changes in body fluids of the cocooning fossorial frog Cyclorana australis in a seasonally dry environment.

We investigated changes in the lymph (equivalent to plasma) and urine of the cocooning frog Cyclorana australis during the dry season in monsoonal northern Australia. Frogs in moist soil for two days were fully hydrated (lymph 220 mOsm kg(-1), urine 49 mOsm kg(-1)). From five weeks onwards the soil was dry (matric potential <-8000 kPa). Aestivating frogs at three and five months formed cocoons in shallow (<20 cm) burrows and retained bladder fluid (25-80% of standard mass). After three months, urine but not lymph osmolality was elevated. After five months, lymph (314 mOsm kg(-1)) and urine (294 mOsm kg(-1)) osmolality and urea concentrations were elevated. Urea was a major contributing osmolyte in urine and accumulated in lymph after five months. Lymph sodium concentration did not change with time, whereas potassium increased in urine after five months. Active animals had moderate lymph osmolality (252 mOsm kg(-1)), but urea concentrations remained low. Urine was highly variable in active frogs, suggesting that they tolerate variation in hydration state. Despite prolonged periods in dry soil, osmolality increase in C. australis was not severe. Aestivation in a cocoon facilitates survival in shallow burrows, but such a strategy may only be effective in environments with seasonally reliable rainfall.

Urinary hormone analysis assists reproductive monitoring and sex identification of bell frogs (Litoria raniformis).

With the world currently facing a global amphibian extinction crisis, the development of techniques to help meet the needs of conservation managers and researchers studying the reproductive biology of amphibians is needed. Here, we developed enzyme immunoassays to measure estrone, testosterone, and progesterone hormone metabolites in the urine of Litoria raniformis, the southern bell frog. Concentrations of urinary estrone, testosterone, and progesterone increased during the breeding season for females (P<0.05). Concentrations of urinary testosterone and progesterone increased for males during the breeding season compared with that for months where no reproductive behaviors were observed (P<0.05). Furthermore, urinary estrone concentrations proved to be a reliable sexing tool for adult frogs, with no overlap between the sexes in 98% of cases, regardless of season. There was no difference in estrone (P=0.204) or testosterone (P=0.485) metabolite concentrations between samples taken immediately upon capture and those taken 12 to 24h later from the same individual. Progesterone metabolite concentrations were lower on Day 2 than upon collection (P=0.004). This is the first study to show that urinary hormone analysis can be a useful technique for reproductive monitoring in an amphibian. Additionally, hormone metabolite measures offer promise as sex identification tools for monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.

Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog.

The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4 x 10(-11) to 10(-6) M ANP or 10(-7) to 3 x 10(-4) M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na+-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5 x 10(-7) M KT-5720 and >10(-5) M H-89), but not by a specific PKG inhibitor (5 x 10(-7) M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na+ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na+ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10(-4) M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na+ absorption of the frog urinary bladder.